Molecular detection of Rickettsia felis in common fleas in Greece and comparative evaluation of genotypic methods

Introduction: Rickettsia felis is the causative agent of flea-borne spotted fever (FBSF), an emerging zoonosis. Although there is evidence of FBSF in Greece, fleas, the classic vectors of R. felis, have not been adequately studied. Thus, the aim of this study was to detect and characterize bacteria of genus Rickettsia and especially R. felis from common fleas parasitizing domestic cats and dogs in Greece and evaluate the efficiency of established molecular techniques. Materials and methods: DNA of flea-pools (samples) by animal-host was investigated by quantitative real-time PCRs (qPCR), and 16S metagenomics (16S). Determination of Rickettsia spp., Rickettsia felis-like organisms (RFLOs), and R. felis was based on a combination of qPCRs targeting gltA and ompB genes, 16S automated metagenomics and manual comparison of 16S sequences for >99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST>99). Information for the animal-hosts was available and statistically analyzed. Results: Among 100 flea-pools, R. felis was detected in 14 samples with a combination of six, five and three assays in 10, two and two samples, respectively. The sensitivity of the assays for Rickettsia genus (16S, and genus specific qPCRs) ranged from 62.5% to 93.8% and the specificity from 65.0% to 100%. R. felis-targeting qPCRs for gltA and ompB demonstrated sensitivity and specificity of 92.9% and 100%, and 100.0% and 87.5%, respectively. 16S metagenomics using the assay software was not able to identify R. felis positive specimens, although manual BLAST>99 did identify the species, but demonstrated sensitivity of 92.9% and specificity of 65.0%. No association of the detection rate of Rickettsia genus or R. felis, with the epidemiological data collected, was identified. Conclusions: These observations suggest the occurrence of R. felis in fleas from pets in Attica, Greece, but PCR and sequencing assays varied considerably in sensitivity and specificity and a consensus methodology for assigning the positivity status is required to be established. © 2020 Elsevier B.V

Evidence of Brucella melitensis DNA in the Microbiome of Ctenocephalides felis from Pet Cats in Greece

Cat fleas (Ctenocephalides felis) are the most prevalent ectoparasites of pet animals with cosmopolitan distribution, obligatory hematophagous, and may prey on humans to receive bloodmeals. We studied the microbiota of 100 flea-pools, containing C. felis, and collected from equal number of cats and dogs in the region of Attica, Greece, including Athens. The 16S metagenomics technique detected Brucella spp. nucleotide sequence that was identified as Brucella melitensis DNA by a real-time PCR, in five flea-pools, corresponding to five cats, one owned and the remaining four stray, residing in semiurban and urban areas, respectively. No definite conclusions can be drawn as to the pathway that led to the presence of B. melitensis in common fleas parasitizing cats. We suspect flea or cat contact with wild rodents, ubiquitous in various environments, which participate in the B. melitensis biology. The proximity of the cats and their fleas with humans and previous observations of flea potential to transmit B. melitensis in laboratory animals warrant a more elaborate research as to the vectorial dynamics, the ecological pathways resulting in pathogen carriage, and the risk for public health. © Copyright 2020, Mary Ann Liebert, Inc., publishers 2020

Molecular detection of Rickettsia felis in common fleas in Greece and comparative evaluation of genotypic methods

Introduction: Rickettsia felis is the causative agent of flea-borne spotted fever (FBSF), an emerging zoonosis. Although there is evidence of FBSF in Greece, fleas, the classic vectors of R. felis, have not been adequately studied. Thus, the aim of this study was to detect and characterize bacteria of genus Rickettsia and especially R. felis from common fleas parasitizing domestic cats and dogs in Greece and evaluate the efficiency of established molecular techniques. Materials and methods: DNA of flea-pools (samples) by animal-host was investigated by quantitative real-time PCRs (qPCR), and 16S metagenomics (16S). Determination of Rickettsia spp., Rickettsia felis-like organisms (RFLOs), and R. felis was based on a combination of qPCRs targeting gltA and ompB genes, 16S automated metagenomics and manual comparison of 16S sequences for >99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST>99). Information for the animal-hosts was available and statistically analyzed. Results: Among 100 flea-pools, R. felis was detected in 14 samples with a combination of six, five and three assays in 10, two and two samples, respectively. The sensitivity of the assays for Rickettsia genus (16S, and genus specific qPCRs) ranged from 62.5% to 93.8% and the specificity from 65.0% to 100%. R. felis-targeting qPCRs for gltA and ompB demonstrated sensitivity and specificity of 92.9% and 100%, and 100.0% and 87.5%, respectively. 16S metagenomics using the assay software was not able to identify R. felis positive specimens, although manual BLAST>99 did identify the species, but demonstrated sensitivity of 92.9% and specificity of 65.0%. No association of the detection rate of Rickettsia genus or R. felis, with the epidemiological data collected, was identified. Conclusions: These observations suggest the occurrence of R. felis in fleas from pets in Attica, Greece, but PCR and sequencing assays varied considerably in sensitivity and specificity and a consensus methodology for assigning the positivity status is required to be established. © 2020 Elsevier B.V

Molecular evidence of a broad range of pathogenic bacteria in ctenocephalides spp.: Should we re-examine the role of fleas in the transmission of pathogens?

The internal microbiome of common cat and dog fleas was studied for DNA evidence of pathogenic bacteria. Fleas were grouped in pools by parasitized animal. DNA was extracted and investigated with 16S metagenomics for medically relevant (MR) bacteria, based on the definitions of the International Statistical Classification of Diseases and Related Health Problems (WHO). The MR bacterial species totaled 40, were found in 60% of flea-pools (N = 100), and included Acinetobacter baumannii, Bacteroides fragilis, Clostridium perfringens, Enterococcus faecalis, E. mundtii, Fusobacterium nucleatum, Haemophilus aegyptius, Kingella kingae, Klebsiella pneumoniae, Leptotrichia buccalis, L. hofstadii, Moraxella lacunata, Pasteurella multocida, Propionibacterium acnes, P. propionicum, Proteus mirabilis, Pseudomonas aeruginosa, Rickettsia australis, R. hoogstraalii, Salmonella enterica, and various Bartonella, Staphylococcus, and Streptococcus species. B. henselae (p = 0.004) and B. clarridgeiae (p = 0.006) occurred more frequently in fleas from cats, whereas Rickettsia hoogstraalii (p = 0.031) and Propionibacterium acnes (p = 0.029) had a preference in fleas from stray animals. Most of the discovered MR species can form biofilm, and human exposure may theoretically occur through the flea-host interface. The fitness of these pathogenic bacteria to cause infection and the potential role of fleas in the transmission of a broad range of diseases should be further investigated. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Molecular evidence of a broad range of pathogenic bacteria in ctenocephalides spp.: Should we re-examine the role of fleas in the transmission of pathogens?

The internal microbiome of common cat and dog fleas was studied for DNA evidence of pathogenic bacteria. Fleas were grouped in pools by parasitized animal. DNA was extracted and investigated with 16S metagenomics for medically relevant (MR) bacteria, based on the definitions of the International Statistical Classification of Diseases and Related Health Problems (WHO). The MR bacterial species totaled 40, were found in 60% of flea-pools (N = 100), and included Acinetobacter baumannii, Bacteroides fragilis, Clostridium perfringens, Enterococcus faecalis, E. mundtii, Fusobacterium nucleatum, Haemophilus aegyptius, Kingella kingae, Klebsiella pneumoniae, Leptotrichia buccalis, L. hofstadii, Moraxella lacunata, Pasteurella multocida, Propionibacterium acnes, P. propionicum, Proteus mirabilis, Pseudomonas aeruginosa, Rickettsia australis, R. hoogstraalii, Salmonella enterica, and various Bartonella, Staphylococcus, and Streptococcus species. B. henselae (p = 0.004) and B. clarridgeiae (p = 0.006) occurred more frequently in fleas from cats, whereas Rickettsia hoogstraalii (p = 0.031) and Propionibacterium acnes (p = 0.029) had a preference in fleas from stray animals. Most of the discovered MR species can form biofilm, and human exposure may theoretically occur through the flea-host interface. The fitness of these pathogenic bacteria to cause infection and the potential role of fleas in the transmission of a broad range of diseases should be further investigated. © 2021 by the authors. Licensee MDPI, Basel, Switzerland